In article <9411142223.AA04169 at calvin.jci.tju.edu>, P_Norton at CALVIN.JCI.TJU.EDU (Dr. Pamela Norton) writes:
> Bionetters,
>> As the subject line says, we have been having terrible problems
> getting a SmaI fragment re-subcloned;
dum-te-dum....
>> I am literally tearing my hair out over this one. Does anyone know
> whether Sma tends to nibble 5' or 3' ends? In other words, would it help if
> we try to refill any nibbled ends before ligation? I'm thinking of
Pam,
SmaI is a B****r of an enzyme for uncontrolled digestion of the blunt ends it
produces. My advice is to digest with a _minimum_ amount of enzyme for the
_minimum_ amount of time - for plasmids that works out at 2 X overdigestion
and 1 h at 25C <---- note the temperature!! Then run some on a gel to check
you've got digestion, and it is best to then do a prep gel to retrieve
digested insert. Don't worry about any that hasn't cut, it's not worth the
hassle in the long run.
And again, I'd use BSA (0.15 mg/ml) and spermidine (2mM) in the rx buffer.
Best of luck,
--
Richard P. Grant
University of Oxford, Nuffield Department of Obstetrics and Gynaecology
John Radcliffe Hospital Maternity Department, Headington
OXFORD OX3 9DU. UNITED KINGDOM.
rpgrant at molbiol.ox.ac.uk <>< FAX +44 1 865 69141